Qingcheng Mao, PhD Department of Pharmaceutics University of Washington

1. Identification of BCRP and CYP3A7 for Glyburide Disposition in the Feto-Placental Unit and Implications for Fetal Toxicity Qingcheng Mao, PhD Department of Pharmaceutics University of Washington

2. Gestational Diabetes Mellitus (GDM) Complicating 5-14% of human pregnancies Defined as “glucose intolerance with first onset during pregnancy” If left untreated, poorly controlled glucose levels result in severe adverse pregnancy outcomes. Mother: preeclampsia, cesarean section, high risk of diabetes Infant: stillbirth, hyperglycemia, macrosomia (giant baby) Disease Pathology: Pregnant women are naturally insulin resistant. Pregnant women with GDM are unable to compensate the degree of resistance, having difficulty to secrete enough insulin (impaired pancreatic beta-cell function) or respond to insulin signaling (decreased insulin sensitivity) or a combination of both.

3. Treatment of GDM with Oral Medications Glyburide (which stimulates insulin production and secretion from beta-cells) and metformin (which increases insulin sensitivity) are being increasingly used to treat GDM: Ease of administration (oral drugs); efficacy comparable to insulin; much lower costs …When pregnant women with GDM are treated with glyburide and their fetuses are exposed to the drug de facto… Glyburide does cross the placenta to the fetus in pregnant women. Umbilical cord plasma glyburide concentrations were ~70% of the maternal plasma concentrations (Hebert et al., Clin Pharmacol Ther 85: 607-614, 2009).

4. Fetal Safety of Glyburide • Neonatal outcomes in the offspring of women with GDM receiving glyburide or insulin were compared in several studies and the data were contradictory. • There are studies showing no significant differences in the % of infants who were large for gestational age (LCA), fetal macrosomia, neonatal hypoglycemia, neonatal intensive care unit admissions, and/or fetal anomalies (Langer et al., 2000; Chmait et al., 2004; Rochon et al., 2006). • There are also studies reporting a trend toward higher incidence of infants with macrosomia or LCA and significantly higher rates of neonatal hypoglycemia in the glyburide group compared to the insulin group (Bertini et al., 2005). • A major concern of glyburide use during pregnancy is how really safe the drug is to the developing fetus, both short-term and long-term? • Therefore, studies investigating the mechanisms that control exposure of glyburide to the fetus are needed.

5. The Placental Barrier Apical side (brush border membrane) facing maternal blood BCRP Basal side (basal membrane) facing fetal compartment BCRP: Breast Cancer Resistance Protein/ABCG2 P-gp: P-glycoprotein/ABCB1 Both are ATP-binding cassette (ABC) transporters. Our previous in vitro transport studies have shown that glyburide is an excellent BCRP substrate. 11

6. Using Bcrp1-/- mice to study fetal exposure to glyburide Dosing (1 mg/kg) wild-type FVB and Bcrp1-/- pregnant mice on gestation day 15 by retro-orbital injection Collecting maternal blood and fetuses at various time points (0.5 -240 min) Determining glyburide concentrations in maternal plasma and fetuses by LC-MS If Bcrp1 limits fetal exposure to glyburide, Bcrp1-/- fetuses will show higher glyburide concentrations and exposure (AUC) than WT fetuses. 6

7. Fetal concentration - time profiles of glyburide in pregnant mice

8. Maternal plasma and fetal AUCs of glyburide (1 mg/kg body weight) ________________________________________________________________________________ WT pregnant Bcrp1-/- pregnant p ________________________________________________________________________ Maternal plasma AUC (g·min/ml) 55.8 +/- 2.7 47.9 +/- 2.3 <0.05 Fetal AUC (ng·min/g fetus) 575.6 +/- 44.6 1108.2 +/- 62.3 <0.01 Fetal/maternal plasma AUC ratio 0.010 0.023 ________________________________________________________________________ Unit of fetal/maternal plasma AUC ratio: [(ng·min/g fetus)/(ng·min/ml)] The fetal/maternal plasma AUC ratio of glyburide in Bcrp1-/- mice was >2 times greater than the ratio in wild-type mice. Bcrp1, the murine homolog of human BCRP, in the mouse placenta limits fetal exposure to glyburide during pregnancy. Human BCRP may play a similar role in pregnant women. 8

9. What happens once glyburide gets to the fetus? • Glyburide is known to be extensively metabolized in adult livers by cytochrome P450 (CYP) enzymes such as CYP3A4, CYP2C9 and CYP2C19. We hypothesize that glyburide also undergoes metabolism in the fetus by CYPs expressed in fetal livers. • HFLMs (rich in CYP3A7) generated a metabolic profile that is different to that by HLMs (rich in CYP3A4 and CYP3A5). • The major CYP known to present in human fetal livers is CYP3A7. Shuster et al. BiochemPharmacol2014

10. Metabolic profiles generated by human fetal liver microsomes and rCYP3A7 were similar According to the metabolic profiles, CYP3A7 is likely the major enzyme responsible for glyburide metabolism in the fetus by human fetal livers. M5 is the primary metabolite in the fetus.

11. CYP3A7 is an effective enzyme for glyburide metabolism CYP3A7 may still be important for glyburide metabolism in human fetal livers despite its 3-times lower catalytic efficiency than CYP3A4. [Clint = Vmax/Km]

12. Correlation between CYP3A7 protein expression and glyburide metabolic activity in individual human fetal livers There is a strong correlation between CYP3A7 protein content (determined by a surrogate peptide-based LC-MS/MS method) and product (16α-OH DHEA) formation activity from a CYP3A7 probe substrate (dehydroepiandrosterone, DHEA) or glyburide depletion activity (Clint) in 16 individual human fetal livers.

13. CYP3A7 protein content strongly correlates with 4’-OH MDZ formation, suggesting that CYP3A7 preferentially catalyzes hydroxylation of midazolam at 4’ position. This is consistent with the fact that the majority of human fetal livers had the 1’-OH/4’-OH MDZ ratio < 1. • Glyburide depletion activity (Clint) strongly correlates with 4’-OH MDZ formation. • These results provide further evidence that CYP3A7 is the major enzyme responsible for glyburide metabolism in human fetal livers.

14. Conclusions and Implications Glyburide can cross the placenta by passive diffusion. Fetal exposure to glyburide may be limited by the efflux transporter BCRP that is highly expressed in human placenta. Once in the fetus, glyburide is metabolized in human fetal livers by CYP3A7. In future studies, it is important to test if the major metabolite M5 is pharmacologically active given the fact that other metabolites, such as M1 and M2b, are known to be pharmacologically active. The results imply that alterations in BCRP and/or CYP3A7 expression or activity (e.g. SNPs, medications/xenobiotics) in the feto-placental unit can affect fetal exposure to glyburide and its metabolite, and ultimately fetal safety of the drug.

15. Acknowledgements • Previous graduate students and postdoctoral fellows: Lin Zhou, Yi Zhang, Honggang Wang, Anshul Gupta, Robert Vethanayagam, XiaokunCai, Zhanglin Ni, Eun-Woo Lee, Sibylle Heidelberger, Diana Shuster • Current lab members: Michael Liao and Lyrialle Han (graduate students); Naveen Neradugomma and Christine C. Gao (postdoctoral fellows); Xaioli Zhao (Visiting Scholar) • NIH Funding NICHD Grant P50HD044404 (2002-2007) NICHD Grant U10HD047892 (2004-2014) NIDA Grant P01DA32507 (2013-2018)

16. School of Pharmacy is part of the University of Washington Health Sciences Center

17. Glyburide Stimulates Insulin Secretion Pancreatic Beta-cell K+ K+ K+ K+ K+ K+ K+ K+ K+ Jack deRuiter. (2003).

19. Additional Evidence that CYP3A7 is the Major Enzyme Responsible for Glyburide metabolism by Human Fetal Livers • It has been shown that the Km of CYP3A4 for 1’-OH MDZ and 4’-OH MDZ formation differ by a magnitude of 10 (2-6 µM vs. 40-60 µM, respectively). • We used 8 µM MDZ in the reactions; therefore, if CYP3A4 was expressed in fetal livers, we might expect preferential formation of 1’-OH MDZ and the corresponding 1’-OH/4’-OH MDZ ratio to be far greater than 1. • However, the metabolite ratio was < or around unity for 12 out of 16 fetal livers, suggesting that CYP3A4 may not be expressed in human fetal livers. • One high ratio of 3.4 came from a fetal liver with the CYP3A5*1/*1 genotype, which is associated with expression of functional CYP3A5. This suggests the likelihood that CYP3A5 is present in some human fetal livers. In fact, CYP3A5 can be detected in some of the fetal livers, but was not quantifiable. 19 19